Prion protein (PrP) underlies a spectrum of age-dependent diseases with no established treatment and devastating human and economical consequences. The existence of transmissible forms separates prion diseases from other neurodegenerative illnesses. The "protein only hypothesis" postulates that an abnormal isoform of PrP (PrP Sc)acts as an infectious agent and propagates its pathological conformation using normal isoform (PrP C) as a substrate. The ultimate direct proof of the "protein only hypothesis', the reconstitution of prpSc in vitro from noninfectious PrP, has not been achieved yet. The proposed research aims at reconstitution of the process of prions conversion in vitro following by bioassay in cultured cells. Three strategies are suggested: (i) manipulating conformational properties of recombinant PrP (rPrP) in vitro; (ii) utilizing permanent non-protein components of prion plaques to assist in vitro folding; and (iii) increasing the susceptibility of cells for prion transmission. The use of cultured neuroblastoma cells instead of animals as a target for transmissible agent allows one to reduce the degree of complexity and to determine factors essential for prion transmission. Several hypotheses proposed by the PI are elaborated into three specific aims: (1) we will determine the capabilities of abnormal beta-sheet rich isoforms of rPrP to induce prion propagation in cultured cells; (2) we will examine intracellular localization and stability of exogenously administered beta-isoformsof rPrP; (3) we will examine whether or not prion replication in calls can be initiated by beta-isoformsof rPrP under glycosylation-inhibiting conditions. To study intracellular localization and stability, immunostaining, FRET, and confocal microscopy will be utilized. Proteinase K digestion, detergent solubility, and immunoconformational assays will be used to detect the formation of nascent PrP Sc isoform in cultured cells. When rPrP-dependent model system of prion propagation is developed, new opportunities will arise for studying detailed mechanisms of transmission and propagation of prions in living cells using non-invasive state-of-the-art biophysical techniques. Such knowledge should lay the foundation for novel molecular and pharmacological approaches of treating transmissible and sporadic prion diseases.